ENVS 1800 Lecture Notes - Lecture 3: Dirty Bit

Medical History:
Ms. PW, a 58 yr old Caucasian woman, is referred by her physician for the development of an exercise prescription. She is 6 yr postmenopausal and has never been on hormone replacement therapy. In 1987, she was diagnosed with hyperthyroidism (overactive thyroid gland), which was treated with radioactive iodine. Since the treatment, Ms. PW has been on synthroid, a drug used when the thyroid gland does not produce enough thyroid hormones. She has mild hypertension, which is regulated by blood pressure–altering medications. Ms. PW is 5 ft 5 in. (165 cm) tall with a weight of 128 lb (58 kg), and her body mass index is in the normal range. She does not follow a regular exercise regimen but reports that she enjoys biking.

Diagnosis:
A few months ago, Ms. PW was diagnosed with osteopenia. Considering her medical history, she is likely to become osteoporotic. Her risk factors are age, sedentary lifestyle, being Caucasian, being petite in stature, loss of menstrual function, and lack of thyroid hormone production.

Exercise Test Results:
She recently underwent an exercise stress test that was normal, but no physiological testing was done other than regular blood chemistries.

Exercise Prescription:
A walking program is surely the best choice because it has the potential of improving not only her cardiorespiratory fitness, but also her bone density. However, Ms. PW is particularly fond of bicycling, and therefore the exercise prescription will incorporate some biking activities, as that is likely to increase her adherence to exercise. These cardiorespiratory activities will start slow and gradually progress over time. Ultimately, her goal would be to walk (3.5 mph [5.6 kph] or faster) or bike 30 to 45 min five times a week. In order to reach this goal, Ms. PW may need to start walking or biking at a slower pace and for a shorter distance, but she should strive to exercise five times a week. After it is determined that she can comfortably handle the speed and distance used for the first 2 wk, these two components should be gradually increased. The best choice is to increase the speed a couple of days a week and increase the distance covered on the other days of the week.
A resistance training program should also be implemented, and may be more beneficial with respect to bone than the cardiorespiratory program. Resistance training should also start slowly, with the frequency beginning at 2 d/wk and moving up to 3 or 4 d/wk. Initially, resistance should be low enough that she can easily complete 12 repetitions of an exercise without undue strain. Exercises that target all the major muscle groups should be incorporated, especially for the legs and back. However, exercises that involve spinal flexion should be avoided. In order to strengthen abdominal muscles, Ms. PW can do leg lifts or modified sit-ups, in which only the head is being lifted off the floor. Upon completion of the first 2 wk, during which the patient does one set of 12 repetitions of each exercise, she can add a second set. This same progression can be used after Ms. PW completes another 2 wk. At this point, the patient should start increasing the intensity by slowly increasing the resistance. An adequate resistance at this point would allow the patient to complete no more than 10 repetitions on the second set. All exercises should be performed with slow, controlled movements. Based on Ms. PW’s medical history, this exercise prescription should be well tolerated and should provide optimal health benefits. Individual cases vary, however, and for a woman who has suffered fragility fractures as a result of osteoporosis, even the minimal exercise described here may not be tolerated initially. Such a patient will need a more gradual, less intense program.

Questions:
1. What other information should be obtained from Ms. PW?

2. What other physiological tests might be recommended?

3. Explain the difference in benefits achieved through cardiorespiratory training versus resistance training for this patient. Why is it always important to incorporate both?

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; “protein purification.ppt”

4. What method did we use to attempt to purify your LepA/EF 4 – GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacI^Q -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacI^Q, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacI^Q gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacI^Q and one contained lacI) that pPEM109 could only be maintained in the lacI^Q containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

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DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg²⁺ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

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DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

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DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 – GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 – GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP – specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 – GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 – GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

You're the resident economic expert of Medicine. The CEO, Jeff, is asking you to complete a project that another person, Shawn, had started working on before he retired. The project is time-sensitive. Jeff explains that Medaline has a manufacturing plant that produces a prescription topical cream called DermaPlus, which is used for treating certain skin conditions. Hospitals and pharmacies are the main buyers of DermaPlus. The market for this cream is extremely competitive. Several other firms produce creams that are almost identical to DermaPlus. Medaline's current share of the market for this type of topical cream is so small that it cannot influence the market price. On the other hand, because Medaline is relatively small compared to the size of the market, it can sell as much of the cream as it likes at the prevailing market price. The plant producing DermaPlus has been operating for a little over three years with the same manufacturing equipment. Currently, there are no plans for upgrading or adding to this equipment. Over the last three years, the price of DermaPlus and related creams has been quite volatile and Medaline has tried to react to the changing price by varying its output level to constantly maximize its monthly profit. To date, Medaline has been able to vary monthly production quite easily by taking advantage of a flexible, none union workforce with a large number of part-time workers. However, the workforce at the DermaPlus plant is just about to be unionized. Once that happens, it will become much more difficult to vary the amount of labour used in the short run and therefore much more difficult to vary the monthly production of DermaPlus. Before he left, Shawn had been asked to estimate the short-run cost functions for the DermaPlus manufacturing plant. The goal was to use this information to determine the profit-maximizing output level and use that information to estimate the optimal size for the newly unionized workforce. Jeff tells you that DermaPlus and related products are just about to come under the umbrella of a new reference-based pricing scheme. Under the new scheme, the government will set the price of DermaPlus and competing creams and review that price every two years. Once the price has been set Medaline and other manufacturers simply have to decide how much of the cream, if any, they want to produce and sell. Unfortunately, although the workforce will be unionized in just over a week, the referenced based price for DermaPlus will not be announced for another two months. Consequently, Medaline has to choose the size of its workforce (and therefore its production capacity) before it knows the price it will get for its product. To reduce the uncertainty about this decision, Jeff recently hired a consultant with expertise in the pharmaceutical industry and reference-based pricing to estimate the price that will be announced for DermaPlus. The consultant estimates that there is a 6% chance that the price will be $50 per unit, a 19% chance that the price will be $100 per unit and a 75% chance that the price will be $150 per unit. This is the best estimate the consultant can provide given the lack of information coming from the government about the issue. After giving you this background information, Jeff asks you to complete the following tasks:

1. Determine the profit-maximizing average monthly production capacity for DermaPlus for each of the possible reference-based prices identified by the consultant. Estimate the expected monthly profit in each case.

2. Recommend an average monthly production capacity for the next 12 months given the uncertainty about the price of DermaPlus. Your recommendation will be used to set the size of the manufacturing plant's unionized workforce. (Note: You simply have to determine the best monthly production capacity for the next 12 months, not the number of workers required.)

3. Write a short report summarizing the results of your analysis and any recommendations. Jeff makes it clear to you that he is a risk-neutral person. You only have a week to complete the analysis, interpret the results, and summarize your findings and recommendations in a brief report. When Jeff makes his recommendations about the structure and format of the report you realize that they are much the same for Assignment 1 that you completed at another company. Finally, Jeff gives you a copy of one of Shawn's spreadsheets. This one contains data that Shawn collected on the variables he thought would be needed to estimate the short-run cost functions for DermaPlus and the firm's profit-maximizing output level. Jeff makes it clear that he has complete confidence in Shawn's technical abilities and professional judgment and tells you to take the information in Shawn's spreadsheet at face value and to use it as a starting point for your analysis.